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Cardiac Ischemia–Reperfusion Injury Under Insulin-Resistant Conditions

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Cardiac Ischemia–Reperfusion Injury Under Insulin-Resistant Conditions Yoshii et al. Cardiovasc Diabetol (2019) 18:85 https://doi.org/10.1186/s12933-019-0889-y Cardiovascular Diabetology ORIGINAL INVESTIGATION Open Access Cardiac ischemia–reperfusion injury under insulin-resistant conditions: SGLT1 but not SGLT2 plays a compensatory protective role in diet-induced obesity Akira Yoshii, Tomohisa Nagoshi* , Yusuke Kashiwagi, Haruka Kimura, Yoshiro Tanaka, Yuhei Oi, Keiichi Ito, Takuya Yoshino, Toshikazu D. Tanaka and Michihiro Yoshimura Abstract Background: Recent large-scale clinical trials have shown that SGLT2-inhibitors reduce cardiovascular events in diabetic patients. However, the regulation and functional role of cardiac sodium–glucose cotransporter (SGLT1 is the dominant isoform) compared with those of other glucose transporters (insulin-dependent GLUT4 is the major isoform) remain incompletely understood. Given that glucose is an important preferential substrate for myocardial energy metabolism under conditions of ischemia–reperfusion injury (IRI), we hypothesized that SGLT1 contributes to cardioprotection during the acute phase of IRI via enhanced glucose transport, particularly in insulin-resistant phenotypes. Methods and results: The hearts from mice fed a high-fat diet (HFD) for 12 weeks or a normal-fat diet (NFD) were perfused with either the non-selective SGLT-inhibitor phlorizin or selective SGLT2-inhibitors (tofoglifozin, ipragli- fozin, canaglifozin) during IRI using Langendorf model. After ischemia–reperfusion, HFD impaired left ventricular developed pressure (LVDP) recovery compared with the fndings in NFD. Although phlorizin-perfusion impaired LVDP recovery in NFD, a further impaired LVDP recovery and a dramatically increased infarct size were observed in HFD with phlorizin-perfusion. Meanwhile, none of the SGLT2-inhibitors signifcantly afected cardiac function or myocardial injury after ischemia–reperfusion under either diet condition. The plasma membrane expression of GLUT4 was sig- nifcantly increased after IRI in NFD but was substantially attenuated in HFD, the latter of which was associated with a signifcant reduction in myocardial glucose uptake. In contrast, SGLT1 expression at the plasma membrane remained constant during IRI, regardless of the diet condition, whereas SGLT2 was not detected in the hearts of any mice. Of note, phlorizin considerably reduced myocardial glucose uptake after IRI, particularly in HFD. Conclusions: Cardiac SGLT1 but not SGLT2 plays a compensatory protective role during the acute phase of IRI via enhanced glucose uptake, particularly under insulin-resistant conditions, in which IRI-induced GLUT4 upregulation is compromised. Keywords: SGLT1, Phlorizin, SGLT2-inhibitor, GLUT4, Insulin resistance, Ischemia–reperfusion injury, Glucose uptake *Correspondence: [email protected] Division of Cardiology, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoshii et al. Cardiovasc Diabetol (2019) 18:85 Page 2 of 14 Background evaluated based on either the mRNA or protein expres- Although fatty acids are the predominant fuel of energy sion from whole heart tissues in the relatively chronic metabolism in the normal adult heart, glucose is an phase. We also recently showed that SGLT1 was highly important preferential substrate under specifc patho- expressed in the plasma membrane fraction of the logical conditions, such as ischemia–reperfusion injury human autopsied heart as well as the murine Langendorf (IRI), as it provides greater efciency for producing high- perfused heart, although the transmembrane protein energy products per oxygen molecule consumed than expression was not signifcantly afected, at least during fatty acids [1–6]. During the acute phase of IRI, under the acute phase of IRI [7]. Subsequently, using a murine conditions of impaired mitochondrial oxidative phospho- Langendorf model, we reported that the non-selective rylation, increased glycolysis becomes a major mecha- SGLT-inhibitor phlorizin perfusion predisposes the heart nism by which the heart maintains ATP generation and to profound IRI, which was associated with a signifcant is critical for the cardiomyocyte survival [2]. Terefore, decrease in the cardiac tissue ATP content, as a result of targeting the acceleration of myocardial glucose utiliza- a reduction in the glucose uptake as well as the glycolytic tion during the acute phase of IRI is a potential therapeu- fux in the heart during ischemia–reperfusion [7]. Tese tic strategy for protecting cardiomyocyte and improving data suggest that cardiac SGLT1 signifcantly contributes the cardiac functional recovery [2, 5, 7, 8]. Tis approach to cardioprotection against IRI by replenishing the ATP may become of particular importance under insulin- stores in ischemic cardiac tissues through promoting glu- resistant conditions, such as diabetes mellitus, in which cose utilization. However, the regulation and functional the glucose utilization is further impaired in response to signifcance of cardiac SGLT1 under insulin-resistant various stimuli, including insulin stimulation as well as conditions, in which the GLUT4 expression is decreased, ischemic insult. remain poorly understood. Glucose utilization is initiated by the uptake of glu- Recent studies have shown that chronic excessive cose via glucose transporters, which appears to be the actions of SGLT1 actually have a detrimental impact rate-limiting step in glycolytic fux in the heart [5, 7, 9]. on the heart [20, 22–24]. Considering that SGLT1 was Glucose transporters are divided into two major families: reported to be chronically over-activated in the diabetic facilitated glucose transporters (GLUTs) and sodium- heart [19, 21, 25], the question remains whether cardiac coupled active transporters (SGLTs) [6, 7]. Among the SGLT1 detrimentally impacts the diabetic myocardium, 12 subtypes of GLUTs conserved in mammals, GLUT1 or exerts a protective efect against ischemic insult as a and GLUT4 appear to be the major glucose transport- compensatory mechanism for compromised GLUT4 ers in the heart, and GLUT4 is thought to be the most under insulin-resistant conditions. abundant, accounting for approximately 70% of glucose To better understand the role and functional sig- transporters [6, 9, 10]. GLUT4 resides mainly in the nifcance of cardiac SGLT1 during IRI under insu- intracellular vesicles under basal conditions and is trans- lin-resistant conditions, we studied the responses of located to the plasma membrane in response to insulin phlorizin-perfused mouse hearts to IRI using high-fat as well as other pathological processes, such as ischemic diet (HFD)-induced obese mice. insult. It was previously reported that GLUT4-mediated enhanced glucose transport represents an essential pro- Methods tective mechanism against IRI and other pathological Animal models stresses [2, 8, 9, 11, 12]. However, it was also reported All animal procedures conformed to the National Insti- that the cardiac GLUT4 expression is decreased under tutes of Health Guide for the Care and Use of Labora- insulin-resistant conditions, such as diabetes, in asso- tory Animals and were approved by the Animal Research ciation with the reduction in glucose uptake, leading to Committee at the Jikei University School of Medicine impaired glucose utilization in the heart [10, 13, 14]. (2016-038C4). Male C57BL/6 mice at 8 weeks of age Although the regulation and the functional roles of were fed either a HFD—consisting of 60% calories from GLUTs in the heart have been intensively investigated in fat, 20% from protein, and 20% from carbohydrates a variety of in vitro and in vivo models [2, 8, 9, 12–15], (D12492; Research Diets, New Brunswick, NJ, USA)—or less is known about the role and functional signifcance a matched control normal-fat diet (NFD)—consisting of of SGLTs in the heart, particularly under insulin-resistant 10% calories from fat, 20% from protein, and 70% from conditions. In the heart, SGLT1, not SGLT2, is consid- carbohydrates (D12450J; Research Diets)—for 12 weeks. ered to be the dominant isoform [16–18]. Previous stud- To investigate the dose efects of SGLT2-inhibitors on ies showed that SGLT1 is highly expressed in the heart, myocardial IRI, 10-week-old male ICR mice that had especially under conditions of ischemia [18, 19], hyper- been fed normal chow (CE-2; CLEA Japan, Tokyo, Japan) trophy [18, 20], or diabetes [19, 21], all of which were were used. Yoshii et al. Cardiovasc Diabetol (2019) 18:85 Page 3 of 14 Glucose and insulin tolerance tests ischemia–reperfusion, as previously described [7, 11]. For the glucose tolerance test, mice were fasted for 16 h, Briefy, hearts were sectioned from apex to base into four anaesthetized (isofurane, 1%, inhalation), and 1 mg/g 2-mm sections. To delineate the infarct, sections were body weight of glucose was injected intraperitoneally incubated in 5% (wt/vol) TTC in PBS at 37 °C for 20 min. (i.p.). For
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